We propose the development of an efficient, non-invasive, automated approach for detection of genomic instability which occurs during the growth of cancerous tissues. This diagnostic assay will identify tumor DNA present in peripheral body fluids such as blood, sputum, urine, or stool. The proposal exploits the use of microsatellite loci to identify loss of heterozygosity (LOH) and microsatellite instability (MI) in cancerous cells. The novelty and advantage of the proposal lies in the selection of optimum loci for analyses (i.e. limiting the effects of PCR artifacts which often interfere with this approach), development of multiplex sets (providing greater assay sensitivity and and higher throughput), adaptation to semi-automated non-radioactive detection formats (providing safer assays), and adaptation to a robotic means of sample preparation and amplification set-up (to reduce the requirement for operator training). In Phase I we will demonstrate all fundamental aspects of the proposal required for later phases, including evaluation of LOH and MI in cancer patient samples using microsatellite multiplex sets, determining stability of extra-cellular genomic DNA in blood samples, and identifying a method of DNA sample preparation which is amenable to automation. In Phase II we evaluate individual loci based on performance characteristics, create comprehensive multiplex sets for microsatellite instability analysis, and automate the sample preparation and PCR-set-up processes for LOH and MI analysis of samples received as blood, plasma and tumor tissues. This will provide all necessary materials to allow product formulation and commercialization in Phase III. PROPOSED COMMERCIAL APPLICATION: Three specific areas provide Promega commercial opportunity totally approximately $45 million in the fifth year following completion of Phase II: (1) DNA-based diagnostic assays for cancer diagnostics, (2) genomic DNA purification systems compatible with manual or automated formats, and (3) application of the same diagnostic systems to identification applications (i.e.forensics and paternity determination).